The alphaviruses include well developed biological weapons and highly virulent, emerging pathogens with the potential for endemic establishment in the United States. Eastern (EEEV) and western (WEEV) equine encephalitis are already native to the US, and cause fatal disease following mosquito or aerosol transmission. Venezuelan equine encephalitis virus (VEEV) causes periodic, massive epidemics of human disease that can also be fatal, including a 1971 Texas outbreak, and is also a highly aerosol-infectious weapon. Chikungunya virus (CHIKV) has for decades been an important but neglected cause of severe, chronic arthritis in Africa and Asia, and recently emerged to cause epidemics involving millions of people on islands off the eastern coast of Africa, and in the Indian subcontinent. Hundreds to thousands of excess deaths involving neurologic disease during these outbreaks suggest that CHIKV may have recently become more virulent. Importations into the Americas via viremic travelers, combined with a 2007 Italian CHIK epidemic initiated by a traveler from India, underscore the severe threat that CHIKV poses to the Americas; the risk for endemic establishment includes both neotropical/subtropical regions inhabited by Aedes aegypti, and temperate areas of the U.S. that are populated by the other efficient vector, Ae. albopictus. Vaccines are needed for all of these alphaviruses, both to prevent their potential impact as biological weapons and, especially for VEEV and CHIKV, to control transmission in endemic locations that puts the U.S. at risk for importation and endemic establishment. We will capitalize on our recent chimeric alphavirus design to optimize vaccine candidates for these 4 viruses, and to test them in rodents and nonhuman primates for attenuation, immunogenicity and efficacy. The specific aims are to: 1) Generate and optimize lead vaccine candidates by combining attenuating mutations designed to modulate the host transcriptional shutoff virulence mechanism, and a novel genetic strategy to eliminate mosquito infectivity; 2) Develop cynomolgus macaques as a nonhuman primate model for CHIKV, and characterize the course of infection in these animals, and; 3) Test the final vaccines in murine and nonhuman primate models for attenuation, immunogenicity, efficacy and virulence stability, and evaluate their environmental safety by testing potential mosquito vectors and reservoir hosts, or models thereof. The resulting vaccines will be ready for final product development and clinical trials.